Neutrophils kill bacteria on extracellular complexes of DNA fibers and bactericidal proteins known as neutrophil extracellular traps (NETs). The NET composition and the bactericidal mechanisms they use are not fully understood. Here, we show that treatment with deoxyribonuclease (DNase I) impairs a late oxidative response elicited by Gram-positive and Gram-negative bacteria and also by phorbol ester. Isoluminol-dependent chemiluminescence elicited by opsonized Listeria monocytogenes-stimulated neutrophils was inhibited by DNase I, and the DNase inhibitory effect was also evident when phagocytosis was blocked, suggesting that DNase inhibits an extracellular mechanism of reactive oxygen species (ROS) generation. The DNase inhibitory effect was independent of actin polymerization. Phagocytosis and cell viability were not impaired by DNase I. Immunofluorescence analysis shows that myeloperoxidase is present on NETs. Furthermore, granular proteins were detected in NETs from Rab27a-deficient neutrophils which have deficient exocytosis, suggesting that exocytosis and granular protein distribution on NETs proceed by independent mechanisms. NADPH oxidase subunits were also detected on NETs, and the detection of extracellular trap-associated NADPH oxidase subunits was abolished by treatment with DNase I and dependent on cell stimulation. In vitro analyses demonstrate that MPO and NADPH oxidase activity are not directly inhibited by DNase I, suggesting that its effect on ROS production depends on NET disassembly. Altogether, our data suggest that inhibition of ROS production by microorganism-derived DNase would contribute to their ability to evade killing.